Understanding Dopamine Receptor Mediated Regulation of Insulin Secretion by Two-Color Spatial Intensity Distribution Analysis

Abstract

Dopamine (DA) has been shown to be a negative regulator of glucose-stimulated insulin secretion from pancreatic β-cells. D2-like receptors that are expressed on the plasma membrane of β-cells mediate the DA effect, but the downstream signaling mechanism(s) by which DA acts is not yet known. These receptors are G-protein coupled and could inhibit insulin secretion through various signaling mechanisms. We know that DA reduces intracellular Ca2+ activity (required for insulin secretion), and electrophysiological data suggests that this effect might be mediated by K+ channels (but not the KATP channel). Therefore, we are particularly interested in the teasing out the signaling pathways that could explain this inhibition. We begin by investigating the potential pathways including inhibition of adenyl cyclase by the Gαi subunit, direct inhibition of Ca2+ channels though the Gβγ subunit, and activation of hyperpolarizing inward rectifying K+ channels through the Gβγ subunit. We expressed recombinant D2-like receptors, G-proteins, and possible effectors tagged with fluorescent proteins in MIN6 murine pancreatic β-cells. We measured the interactions of D2-like receptors and G-proteins with possible effectors using Spatial Intensity Distribution Analysis (SpIDA). SpIDA is spectroscopic tool for investigating interactions of fluorescent molecules by analysis of confocal laser scanned microscopy (CLSM) images. We have used an extension of SpIDA to analyze CSLM images in two detection channels. Two-color analysis allows us to quantitatively measure heteromeric interactions with high sensitivity. Preliminary results show minimal DA-induced changes of cAMP levels, suggesting that the Gβγ subunit is the critical signaling molecule facilitating the DA-effect. Data show that DA stimulation disrupts interactions between D2-like receptors and the heterotrimeric G-proteins, but not interactions between Ca2+ channels and the Gβγ subunit, suggesting that the important target is a K+ channel.